The anterior surface of the eye functions as a barrier to the external environment and protects the delicate underlying structures from injury. This protection is provided, in part, through the elaboration of the corneal, limbal and conjunctival epithelia. As self-renewing tissues, these epithelia are governed by stem cells, which play a crucial role in tissue homeostasis, regeneration, tissue transplantation, gene therapy and in the pathogenesis of several anterior ocular surface diseases. It is well-accepted that that the corneal epithelial stem cells are primarily located in the basal layer of the limbal epithelium. Despite the many advances made in our understanding of limbal epithelial stem cell biology, major questions remain unresolved concerning stem cell maintenance. Specifically: (i) what keeps stem cells in their quiescent state yet enables these cells to periodically divide to give rie to TA cells; and (ii) how do these stem cells regulate their energy metabolism? Towards this end, we have recently discovered that the hydroxylase, factor inhibiting hypoxia-inducible factor- 1 (FIH-1), is highly expressed in human limbal epithelium, but not in central corneal epithelium. Furthermore, we have evidence that FIH-1 functions on two levels in cultured human corneal epithelial keratinocytes (HCEKs): (i) it blocks Notch-dependent corneal epithelial differentiation; and (ii) it decreases glycogen stores most likely through Akt signaling. Given the importance of these two processes to corneal epithelial maintenance, we propose to test our hypothesis that FIH-1, a heretofore unrecognized component of limbal epithelium, is a pleiotropic hydroxylase with major consequences for limbal epithelial stem cell homeostasis. Towards this end, we will focus on how FIH-1: (i) maintains stemness by regulating Notch activity; and (ii) regulates limbal and corneal epithelial glycogen metabolism. To accomplish these goals we will capitalize on our ability to modulate FIH-1 levels in cultured human limbal epithelial keratinocytes (HLEKs) and HCEKs, three-dimensional organotypic raft cultures of limbal and corneal epithelia, and mice and assess the functional consequences of such modulation with a combination of biochemical, molecular biological, cell biological and physiological approaches. Information from these studies will provide insight into how FIH-1 regulates limbal and corneal epithelial development, differentiation and physiology. By focusing on the biology of FIH-1, our application represents a new approach to study: (i) how the transition from proliferation to differentiation is regulated; and (ii) what contributes to the maintenance of cellular glycogen. Such knowledge has relevance not only to stem cell biology but to disorders of corneal epithelial differentiation, particularly:(i) dystrophies; (ii) dry eye;(iii) keratitis resulting from excessive glycogen depletion; and (iv) diabetic-induced keratopathies. Understanding the biology of FIH-1 will provide a rationale for the development of innovative treatment regimens focused on the use of inhibitors of this hydroxylase in patients with diseases such as these that affect the ocular anterior segmental epithelia. PUBLIC HEALTH RELEVANCE: Information from this project will impact on our understanding of how the hydroxylase, factor inhibiting hypoxia-inducible factor-1 (FIH-1) contributes to the maintenance of corneal epithelial stem cells. These studies will lead to a better comprehension of disorders of corneal epithelial differentiation, particularly:(i) dystrophies; (ii) dry eye; (ii) keratitis resulting from excessive glycogen depletion; and (iv) diabetic-induced keratopathies. A benefit from this research will be the development of innovative treatment regimens focused on the modulation of FIH-1 in patients with diseases affecting the corneal epithelium.